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Structural Basis of Salicylic Acid Decarboxylase Reveals a Unique Substrate Recognition Mode and Access Channel
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2021-09-23 , DOI: 10.1021/acs.jafc.1c04091 Xin Gao 1 , Mian Wu 1 , Wei Zhang 1 , Chao Li 1 , Rey-Ting Guo 2 , Yujie Dai 1 , Weidong Liu 3 , Shuhong Mao 1 , Fuping Lu 1 , Hui-Min Qin 1
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2021-09-23 , DOI: 10.1021/acs.jafc.1c04091 Xin Gao 1 , Mian Wu 1 , Wei Zhang 1 , Chao Li 1 , Rey-Ting Guo 2 , Yujie Dai 1 , Weidong Liu 3 , Shuhong Mao 1 , Fuping Lu 1 , Hui-Min Qin 1
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Salicylic acid (SA) decarboxylase from Trichosporon moniliiforme (TmSdc), which reversibly catalyses the decarboxylation of SA to yield phenol, is of significant interest because of its potential for the production of benzoic acid derivatives under environmentally friendly reaction conditions. TmSdc showed a preference for C2 hydroxybenzoate derivatives, with kcat/Km of SA being 3.2 × 103 M–1 s–1. Here, we presented the first crystal structures of TmSdc, including a complex with SA. The three conserved residues of Glu8, His169, and Asp298 are the catalytic residues within the TIM-barrel domain of TmSdc. Trp239 forms a unique hydrophobic recognition site by interacting with the phenyl ring of SA, while Arg235 is responsible for recognizing the hydroxyl group at the C2 of SA via hydrogen bond interactions. Using a semi-rational combinatorial active-site saturation test, we obtained the TmSdc mutant MT3 (Y64T/P191G/F195V/E302D), which exhibited a 26.4-fold increase in kcat/Km with SA, reaching 8.4 × 104 M–1 s–1. Steered molecular dynamics simulations suggested that the structural changes in MT3 relieved the steric hindrance within the substrate access channel and enlarged the substrate-binding pocket, leading to the increased activity by improving substrate access. Our data elucidate the unique substrate recognition mode and the substrate entrance tunnel of SA decarboxylase.
中文翻译：
水杨酸脱羧酶的结构基础揭示了独特的底物识别模式和访问通道
来自串珠孢子虫 ( TmSdc ) 的水杨酸 (SA) 脱羧酶可逆地催化 SA 脱羧生成苯酚，因其在环境友好的反应条件下生产苯甲酸衍生物的潜力而备受关注。TmSdc 表现出对 C2 羟基苯甲酸酯衍生物的偏好， SA 的k cat / K m为 3.2 × 10 3 M –1 s –1. 在这里，我们展示了 TmSdc 的第一个晶体结构，包括与 SA 的复合物。Glu8、His169 和 Asp298 的三个保守残基是 TmSdc 的 TIM 桶结构域内的催化残基。Trp239 通过与 SA 的苯环相互作用形成独特的疏水识别位点，而 Arg235 负责通过氢键相互作用识别 SA 的 C2 上的羟基。使用半理性组合活性位点饱和测试，我们获得了 TmSdc 突变体 MT3 (Y64T/P191G/F195V/E302D)，其k cat / K m与 SA 增加了 26.4 倍，达到 8.4 × 10 4 M –1秒–1. 转向分子动力学模拟表明，MT3 的结构变化减轻了底物进入通道内的空间位阻并扩大了底物结合袋，从而通过改善底物进入提高了活性。我们的数据阐明了 SA 脱羧酶独特的底物识别模式和底物入口通道。
更新日期：2021-10-06
中文翻译：
水杨酸脱羧酶的结构基础揭示了独特的底物识别模式和访问通道
来自串珠孢子虫 ( TmSdc ) 的水杨酸 (SA) 脱羧酶可逆地催化 SA 脱羧生成苯酚，因其在环境友好的反应条件下生产苯甲酸衍生物的潜力而备受关注。TmSdc 表现出对 C2 羟基苯甲酸酯衍生物的偏好， SA 的k cat / K m为 3.2 × 10 3 M –1 s –1. 在这里，我们展示了 TmSdc 的第一个晶体结构，包括与 SA 的复合物。Glu8、His169 和 Asp298 的三个保守残基是 TmSdc 的 TIM 桶结构域内的催化残基。Trp239 通过与 SA 的苯环相互作用形成独特的疏水识别位点，而 Arg235 负责通过氢键相互作用识别 SA 的 C2 上的羟基。使用半理性组合活性位点饱和测试，我们获得了 TmSdc 突变体 MT3 (Y64T/P191G/F195V/E302D)，其k cat / K m与 SA 增加了 26.4 倍，达到 8.4 × 10 4 M –1秒–1. 转向分子动力学模拟表明，MT3 的结构变化减轻了底物进入通道内的空间位阻并扩大了底物结合袋，从而通过改善底物进入提高了活性。我们的数据阐明了 SA 脱羧酶独特的底物识别模式和底物入口通道。
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